No multiplication of the virus in vitro was observed. We have found, however, that the optimum conditions necessary for the preservation of the virus in artificial media are as follows:
The hydrogen ion concentration of the medium should be 7.5 to 7.6, not only at the beginning, but, and more important, at the conclusion of the period of observation. A strict anaerobic atmosphere is also favorable, as is a temperature below 37°C. A semisolid structure of the medium appears to be advantageous and this can be effected by the use of ¼th per cent agar or 10 per cent gelatin. Of the two, the gelatin is more desirable, and of the latter the most effective material is gelatin from which the impurities have been most thoroughly removed, namely the gelatin employed by Loeb for his isoelectric determinations. This material is best adjusted to the proper hydrogen ion concentration (7.5–7.6) with potassium hydroxide and not with buffer phosphate.
Gelatin is the simplest of protein media available and its employment is in keeping with the principle we have found that the requirements for life of the virus of foot-and-mouth disease are of the simplest. The addition of organic or complex protein substances, such as dextrose, broth, serum, lipoids, etc., to a simple basic medium interferes with the effectiveness of the latter. It is thus not surprising that we were unable to confirm the cultural results of Frosch and Dahmen for neither their medium nor its components or their method satisfies the essential conditions necessary to maintain artificially the life of the virus.
Furthermore, from the standpoint of technique it was found necessary when comparing 2 or more media for their value in preserving the incitant, to employ all of them in a parallel experiment with the same sample of virus, for the factors of potency of the active agent, contamination, and changes in hydrogen ion concentration, if variable, may give rise to faulty interpretations. Moreover, activity in 3 successive subplants may be regarded as mere preservation but not multiplication of the virus.
Finally, we discuss the status of the virus from the point of view of its fluid or particulate, and its animate or inanimate, characters.